The effect of chitosan on stiffness and glycolytic activity of human bladder cells.

The cell's cytoskeleton together with the cell membrane and numerous accessory proteins determines the mechanical properties of cell. Any factors influencing cell organization and structure can cause alterations in mechanical properties of cell (its ability for deformation and adhesion). The determination of the local elastic properties of cells in their culture conditions has opened the possibility for the measurement of the influence of different factors on the mechanical properties of the living cells. The effect of the chitosan on the stiffness of the non-malignant transitional epithelial cells of ureter (HCV 29) and the transitional cell cancer of urine bladder (T24) was determined using scanning force microscopy. The investigations were performed in the culture medium (RPMI 1640) containing 10% fetal calf serum in the presence of the microcrystalline chitosan of the three different deacetylation degrees. In parallel, the effect of chitosan on production of lactate and ATP level was determined. The results showed the strong correlation between the decrease of the energy production and the increase in Young's modulus values obtained for the cancer cells treated with chitosan.

Amino-acid composition of pyruvate kinase M2 isoenzyme variants from rat liver and Morris hepatoma 7777.

Cytosolic fractions B (salted out between 51-70% ammonium sulphate saturation) from rat liver and Morris hepatoma 7777, containing pyruvate kinase (EC 2.7.1.40) M2 isoenzymes, were purified by affinity chromatography on Blue Sepharose CL-6B. When compared by polyacrylamide gel electrophoresis at pH 8.3, all three M2 pyruvate kinase variants from Morris hepatoma 7777 had lower mobilities (alpha2, beta2, gamma3) than the three corresponding variants (alpha1, beta1, gamma2) from normal rat liver. Using an automatic amino-acid analyser, significant differences in selected amino-acid content have been found in corresponding highly purified gamma3 and gamma2 variants from Morris hepatoma and normal rat liver, respectively. The gamma3-variant of the Morris hepatoma M2 isoenzyme had twice the amount of L-tyrosine and L-cysteine, and a content of L-serine higher by 20% than the corresponding gamma2 variant of the normal rat liver M2 isoenzyme. It contained, however, significantly less dicarboxylic amino acids which explains its lower electrophoretic mobility. It showed also a decrease (by about 10%) in several other amino-acid content, corresponding to a 10% decrease in the tumour enzyme molecular mass.

N-acetylneuraminic acid, phosphate and thiol groups of pyruvate kinase isoenzymes from Morris hepatoma 7777 and normal rat liver.

The highest amount of N-acetylneuraminic acid (AcNeu) was found in pyruvate kinase isoenzyme L from normal rat liver (24 moles/mole of enzyme tetramer), with the highest electrophoretic mobility. On the other hand, isoenzyme M2 from Morris hepatoma 7777, with the lowest electrophoretic mobility, had the lowest AcNeu content (5 moles/mole of enzyme tetramer). This tumour isoenzyme M2 of pyruvate kinase was, however, characterised by the highest phosphate content (12 moles/mole protein), in comparison to isoenzyme L (3 moles/mole protein) or normal liver isoenzyme M2 (6 moles/mole protein). This could indicate a regulatory change caused by reversible enzyme phosphorylation and dephosphorylation or sialization and desialization. Despite these differences, the sum of the two negatively charged residues was lower in tumour pyruvate kinase isoenzyme M2, with the slowest migration rate, than in normal rat liver isoenzyme M2. Moreover, isoenzyme M2 from tumour material, in comparison with isoenzyme M2 from normal rat liver, had a twice as high content of thiol groups (20 moles/mole protein), especially of free and superficially located ones, than the isoenzyme M2 from normal liver (10 moles/mole protein). This may explain abnormal susceptibility of tumour isoenzyme M2 to stereospecific inhibition by exogenous L-cysteine, and indicate genetically dependent changes in amino-acid content of tumour enzyme which take place during cell tumourigenic transformation.

Tumor-specific pyruvate kinase isoenzyme M2 involved in biochemical strategy of energy generation in neoplastic cells.

The differences in properties of pyruvate kinase (EC 2.1.7.40) from normal tissues and animal or human tumors are described and their significance for various metabolic abnormalities is reviewed. The tumor variant gamma3 from M2 isoenzyme of pyruvate kinase sensitive to L-cysteine inhibition, when over-expressed, can be used as a marker of tumorigenic transformation. It seems that this variant represents a tumor-specific oncoprotein, involved in a novel metabolic strategy of energy generation during increased cell proliferation.

In vitro inhibitory effect of chitosan and its degradation products on energy metabolism in Ehrlich ascites tumour cells (EAT).

Chitosan, a product of depolymerization and deacetylation of chitin, as polycation, diminishes glycolysis, i.e. aerobic lactate formation both, in Ehrlich ascites tumour (EAT) intact cells and in their cytosolic fraction. By inhibiting glycolysis, chitosan decreases also glucose uptake and ATP level in intact cells. Chitosan does not exert a similar inhibitory effect on glycolytic activity of mouse normal liver and muscle supernatants. It has been found that a decrease in glycolytic activity and cell ATP level in tumour cells is caused by inhibition of tumour specific variant of pyruvate kinase from fraction B (isoenzyme M2).

Electrophoretic pattern of cytosolic pyruvate kinase fractions A and B (type L and M2) from normal rat liver and Morris hepatoma 7777.

Cytosolic pyruvate kinase fractions A and B obtained by salting out procedure from normal rat liver and Morris hepatoma 7777, purified by affinity chromatography on Blue Sepharose CL-6B, have shown similar electrophoretic patterns in polyacrylamide gel at pH 8.3 to previously studied pyruvate kinase extracts from chromatin of cell nuclei. Three variants (alpha 1, beta 1, gamma 1) from normal rat liver pyruvate kinase fraction A (type L) had the greatest electrophoretic mobility, showed sigmoidal kinetics in relation to 2-phosphoenolpyruvate (PEP), and sensitivity to ATP and fructose 1,6-diphosphate (FDP). The fraction A dominated over normal liver fraction B (type M2), which in electrophoresis showed a slower gamma 2 variant, similar to the fraction A of hepatoma. All variants from fractions B of normal liver and A of hepatoma had linear kinetics and were sensitive to ATP but not to FDP. The greatest differences showed pyruvate kinase fraction B from Morris hepatoma. Its all variants alpha 2, beta 2, gamma 3 were cathodic and had linear kinetics in relation to PEP. They all were insensitive to normal signal molecules (ATP and FDP). The gamma 3 alkaline variant acquired sensitivity to inhibition by L-cysteine. Showing several-fold higher activity, much greater affinity to the main substrate, and a lack of sensitivity to feed-back inhibition by ATP, it was responsible for a high rate of aerobic glycolysis and diminution of the Pasteur effect in metabolic studies. It was probably encoded during oncogene activation and plays a special role in different metabolic strategies of tumour cells.

Comparison of pyruvate kinase variants from rat liver and Morris hepatoma 7777, obtained by an affinity chromatography on blue sepharose CL-6B.

Fractions A (salted out by ammonium sulphate between 21-30% saturation), and fractions B (salted out between 51-70% saturation) of pyruvate kinase (EC 2.7.1.40.) corresponding respectively to pyruvate kinase types L and M2 from rat liver and Morris hepatoma 7777 were purified by an affinity chromatography on Blue Sepharose CL-6B. Peaks of inactive proteins were eliminated and the enzyme fractions bound biospecifically to the gels were eluted by free ADP. The molecular mass of purified hepatoma pyruvate kinase fraction B was smaller than that of liver pyruvate kinase fraction B. Morris hepatoma pyruvate kinase fraction B represented a variant of type M2, characterised by greatest affinity to 2-phosphoenolpyruvate as a main substrate and different sensitivity to low-molecular effectors in comparison with types L from both liver and hepatoma and in comparison with type M2 from normal rat liver. Only this hepatoma fraction B showed a tumour specific sensitivity to L-cysteine and was insensitive to normal signal molecules i.e. to ATP and fructose-1,6-diphosphate which influence liver pyruvate kinase activity. L-Cysteine inhibited the tumour fraction B of pyruvate kinase by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate.

Effect of environmental fluorides on key biochemical processes in humans.

Several biochemical parameters connected with energy metabolism were studied in the inhabitants living in the neighbourhood of the Aluminium Smelter and in inhabitants of Chorzów, one of the most polluted towns in the Upper Silesian Industrial Region, chronically exposed to environmental fluorides and other toxic compounds. In people exposed to environmental fluorides excessive excretion of fluorides in urine was noted. This effect was connected with a decrease in urine and erythrocyte magnesium concentrations. Among several biochemical parameters an increase in blood glucose and lactate levels were found, leading to a decrease in blood ATP level. Supplementation of magnesium salts in inhabitants of Skawina caused a normalization of urine magnesium concentration, blood glucose and lactate levels, and increased blood ATP level, but did not diminish fluoride excretion in urine.

[Selected biochemical indicators of the effect of environmental lead on the humans]. / Wybrane biochemiczne wskazniki oddzialywania olowiu srodowiskowego na ludzi.

Lead after penetration into the human organism reacts with different sulphur compounds, especially with thiol group of peptides and proteins. It diminishes haeme synthesis by inhibiting thiol enzymes of bone marrow. Measurements performed in three groups of inhabitants of Chorzów--the most polluted town in the Upper Silesia, revealed in approx. 70% of workers of Nitrogen Chemical Plant (group A) and Steelworks (group S) a several-fold higher concentrations of blood lead, than in workers of Slaughterhouse (group M) in which it remained within the normal range for urban populations. In all subjects examined, the level of reduced glutathione in erythrocytes was approx. 50% lower in comparison to normal mean value. In urine, the highest excretion of delta-aminolevulinic acid (ALA), a precursor for haeme synthesis was found in group A, characterized by highest blood lead as well as in group M, with normal blood lead concentrations. The ALA excretion in group S was lower but it also exceeded the upper limit of normal range. Amounts of porphobilinogen excreted in urine exceeded the normal range, more frequently in group A and M than in group S, as in the case of ALA. These results indicate a widespread exposition of Chorzów inhabitants to lead, although the altered amounts of compounds studied show no correlation with the lead concentrations in blood. It seems that blood lead concentration may be modified depending on individual metabolic features, work conditions or diet.

[Fluorides in urine of the population living in polluted environment]. / Fluor w moczu u ludzi zyjacych w zanieczyszczonym srodowisku.

The increased excretion of fluorides in urine was shown by ion-selective method, in three different groups of workers, inhabitants of Chorzów--the most polluted town in the Upper Silesia. In nearly all workers (90%) of Nitrogen Chemical Plant and Slaughterhouse (groups A and M), the urine fluoride exceeded three to four times the upper limit of the normal range, while only two times in slightly less number of workers (77%) of the Steelworks (group S). The subjective complaints and bone pains were correlated with excessive urine fluoride concentrations in about 40% of workers studied in groups A and M, and in 18% of workers in group S. These results point to an excessive exposition of people to fluorides from the polluted environment, as well as to a high risk of fluorine retention in bones and osteofluorosis.

[Inhibitory effect of environmental pollutants on erythrocyte membrane ATPase activity in humans]. / Hamujacy wplyw zanieczyszczen srodowiskowych na aktywnosc ATPaz z blon erytrocytów ludzi.

In the inhabitants of Chorzów, the most polluted town in the Upper Silesia, who showed increased urine fluoride excretion and elevated blood lead concentrations, the studies on erythrocyte membrane ATPase activities have been undertaken. The investigations showed the decrease in (Na,K)ATPase activity by about 70% in comparison with the average norm in 85% of workers of the Nitrogen Chemical Plant and the Slaughterhouse (groups A and M) and by about 50% in 65% of workers of the Steelworks (group S). The decrease in Mg-ATPase activity was smaller, by about 40% in 70% persons in groups A and M, and only by 20% in comparison with the control activity in 40% persons in group S. The correlation between the decrease in (Na,K)ATPase activity in vivo and the increase in urine fluoride excretion was demonstrated. Although the possibility of lead influence on this enzyme in vivo cannot be excluded, the correlation between the changes in ATPase activity and blood lead concentration was not found.

Pyruvate kinase isoenzymes in chromatin extracts of Ehrlich ascites tumour, Morris hepatoma 7777 and normal mouse and rat livers.

A pyruvate kinase (EC 2.7.1.40) variant inhibited by L-cysteine has been found in Ehrlich ascites tumour and Morris hepatoma 7777, but not in normal mouse and rat livers used for comparison. Chromatin extracts of all materials studied contained three pyruvate kinase isoenzymes (alpha, beta, gamma) which showed the greatest electrophoretic mobility in normal mouse and rat livers. The isoenzyme mobility diminished in both tumour chromatin extracts, and the slow migrating gamma isoenzyme exhibited sensitivity to L-cysteine inhibition. This gamma isoenzyme sensitive to L-cysteine might be considered as a tumour marker. All tumour pyruvate kinase isoenzymes were insensitive to normal signal molecules, i.e., to ATP and fructose 1,6-diphosphate, which regulate liver pyruvate kinase activity. It was, however, noted that the binding of pyruvate kinase isoenzymes to DNA is connected with a diminution in their catalytic activity.

Metabolism of cysteine in Ehrlich ascites tumour, mouse skeletal muscles and liver.

The cell membrane of intact Ehrlich ascites tumour (EAT) cells limits the penetration and thus the utilization of exogenous cysteine in vitro. In homogenates the uptake of cysteine is the greatest in skeletal muscle, smaller in EAT and the smallest in liver. 2-Oxoglutarate, initiating the pyruvate pathway of cysteine catabolism, raises cysteine utilization only in liver homogenates. Although it also raises taurine formation, it shifts the equilibrium from the oxidative toward the anaerobic pyruvate pathway of cysteine metabolism. 2-Oxoglutarate has no effect on cysteine metabolism in EAT and only a small effect on this process in muscle homogenates. Limitation of cysteine metabolism in the pyruvate pathway in tumour cells is not compensated by increased cysteine oxidation. The greatest increase in protein thiol groups was observed in EAT homogenates; it was markedly reduced in the presence of 2-oxoglutarate.